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| DC 欄位 | 值 | 語言 |
|---|---|---|
| dc.contributor.advisor | 蔡素宜(Su-Yi Tsai) | |
| dc.contributor.author | Chia-Wei Chang | en |
| dc.contributor.author | 張家維 | zh_TW |
| dc.date.accessioned | 2021-06-17T06:14:36Z | - |
| dc.date.available | 2019-09-25 | |
| dc.date.copyright | 2018-09-25 | |
| dc.date.issued | 2018 | |
| dc.date.submitted | 2018-09-17 | |
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| dc.identifier.uri | http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/71910 | - |
| dc.description.abstract | 心臟肌節基因的缺陷通常造成嚴重的心肌疾病，而目前大多數治療心臟衰竭的方法無法根治肌肉缺陷的問題。RBM24被視作為調節心臟發育及肌節發育過程中，調控mRNA拼接的重要角色。然而，RBM24中具有主要功能的區域及其在人類心肌細胞發育過程中詳細的功能仍未被證實。本研究在人類胚胎幹細胞中利用CRISPR/Cas9系統分別剔除RBM24基因之兩個不同區域，以測試RBM24之RRM 結構域的功能。在此結果中，兩種剔除細胞株之心肌細胞雖然可以正常跳動，但剔除RRM結構域之細胞株之心肌細胞呈現肌節構造排列不規則及不正常之粒線體表現型。對比之下，於Exon2剔除細胞株在肌節構造、心肌細胞大小及粒線體形態上均呈正常表現型。整體而言，本研究不只初步證明了RBM24之RRM結構域在人類胚胎幹細胞衍生之心肌肌節生成中扮演必要角色，並更進一步發現RBM24蛋白可能影響心肌細胞中粒線體之功能。未來我將利用RNA sequencing的方法，去更進一步了解RRM結構域之功能在心肌肌節生成的分子機制。 | zh_TW |
| dc.description.abstract | The abnormal expression of cardiac sarcomere genes usually results in cardiomyopathy. However, current treatments for heart failure do not address the root problem involving cardiac muscle deficiencies. RNA-binding motif protein 24 (RBM24) is a key regulator of the alternative splicing of mRNA during cardiomyogenesis and sarcomerogenesis. The functional region of RBM24 that mediates cardiac development in humans remains to be elucidated. In this thesis project, I used human embryonic stem cells (hESCs) as a model system and eliminated two RBM24 regions using the CRISPR/Cas9 system to functionally characterize the RBM24 RRM domain. Although cardiomyocytes (CMs) derived from two types of mutant lines were still able to induce a normal heartbeat, CMs derived from the ∆RRM-/- mutants exhibited a disorganized sarcomeric structure and abnormal mitochondrial morphology. In contrast, the ∆Exon2-/- mutants produced a well-organized sarcomeric structure, with a normal CM size and mitochondrial structure. Considered together, the data presented herein reveal that the RBM24 RRM domain is essential for ensuring a normal sarcomeric structure in hESC-derived CMs. These findings not only represent some evidence of the importance of the RBM24 RRM domain, they also suggest that RBM24 may regulate mitochondrial functions in human CMs. Future experiments will involve the application of RNA sequencing to further characterize the molecular mechanism underlying the effects of the RRM domain on sarcomerogenesis. | en |
| dc.description.provenance | Made available in DSpace on 2021-06-17T06:14:36Z (GMT). No. of bitstreams: 1 ntu-107-R05b21028-1.pdf: 20097862 bytes, checksum: 82e377a5bbb9e695920a7f69c39a8450 (MD5) Previous issue date: 2018 | en |
| dc.description.tableofcontents | 論文口試委員審定書 i
致謝 ii 中文摘要 iii Abstract iv Introduction 1 1. Application of human pluripotent stem cells 1 2. Sarcomerogenesis 2 3. Overview of RNA-binding motif protein 24 3 3.1 RNA-binding motif proteins 3 3.2 Introduction of RNA-binding motif protein 24 5 Materials and Methods 8 Human embryonic stem cell culture and Targeting strategy 8 Transfection of CRISPR/Cas9 plasmid and SURVEYOR assay 9 Western blot 10 Cardiac differentiation 10 Immunofluorescence staining 11 TEM samples preparation and fixation 12 Statistical analysis 13 Results 14 Elimination of RBM24 in human embryonic stem cell 14 Pluripotency and cellular differentiation in the two types of RBM24 knockout lines 15 Sarcomere structure of RBM24 mutant cardiomyocytes 16 Sizes of cardiomyocytes and nuclei in RBM24 mutant lines 18 Proliferation rate of RBM24 mutant cardiomyocytes 19 Knockout of the RRM domain of RBM24 might affect mitochondria in cardiomyocytes 19 Discussion 21 References 23 Tables &Figures 31 Table 1. sgRNA primers 31 Table 2. RBM24 PCR primers for genotyping 32 Table 3. Antibodies used for immunostaining/western blot 33 Table 4. Amino acid sequence prediction 35 Figure 1. Elimination different region of RBM24 in hESCs by using CRISPR/Cas9 system. 37 Figure 2. Elimination different region of RBM24 has no effect on pluripotent markers NANOG and OCT4. 38 Figure 3. RBM24 mutant lines expressed MYH6:mCherry after differentiation into cardiomyocytes. 39 Figure 4. Z-line structure of RBM24 mutant cardiomyocytes. 41 Figure 6. The transmission electron microscopy images of sarcomere structure. 45 Figure 7. Quantification of cardiomyocyte size in the RBM mutant lines. 46 Figure 8. Mitochondrion morphology in mutant cardiomyocytes. 47 Figure 9. The proliferation rate of RBM24 mutation cardiomyocyte. 49 Supplemental Tables &Figures 50 Table S1. Raw data of cardiomyocyte cell size 50 Table S2. Raw data of cardiomyocyte nuclei size 51 Table S3. Raw data of cardiomyocyte proliferation rate 53 Figure S1. SURVEYOR assay for testing RBM24 SgRNA targeting efficiency. 54 Figure S2. Genotyping results of RBM24 mutant lines. 55 Figure S3. The organized z-disc structure presented on differentiation day30 RRM domain elimination cardiomyocytes. 56 Figure S4. Instruction of cell size and nucleus size. 57 | |
| dc.language.iso | en | |
| dc.subject | RNA結合模組蛋白24 | zh_TW |
| dc.subject | CRISPR/Cas9系統 | zh_TW |
| dc.subject | 心臟性分化 | zh_TW |
| dc.subject | 肌節生成 | zh_TW |
| dc.subject | 心肌細胞 | zh_TW |
| dc.subject | 人類全能幹細胞 | zh_TW |
| dc.subject | RRM結構域 | zh_TW |
| dc.subject | human pluripotent stem cell | en |
| dc.subject | sarcomerogenesis | en |
| dc.subject | CRISPR/Cas9 system | en |
| dc.subject | RRM domain | en |
| dc.subject | cardiomyocyte | en |
| dc.subject | RNA-binding motif protein 24 | en |
| dc.subject | cardiac differentiation | en |
| dc.title | 利用人類多能胚胎幹細胞衍生之心肌細胞探討RBM24之功能 | zh_TW |
| dc.title | Functional Study of RNA-Binding Motif Protein 24 in Human Pluripotent Stem Cell-Derived Cardiomyocytes | en |
| dc.type | Thesis | |
| dc.date.schoolyear | 107-1 | |
| dc.description.degree | 碩士 | |
| dc.contributor.oralexamcommittee | 陳彥榮(Yan-Rung Chen),李士傑(Shyh-Jye Lee),陳文彬(Wen-Pin Chen) | |
| dc.subject.keyword | 人類全能幹細胞,RNA結合模組蛋白24,RRM結構域,心肌細胞,肌節生成,心臟性分化,CRISPR/Cas9系統, | zh_TW |
| dc.subject.keyword | human pluripotent stem cell,RNA-binding motif protein 24,RRM domain,cardiomyocyte,sarcomerogenesis,cardiac differentiation,CRISPR/Cas9 system, | en |
| dc.relation.page | 57 | |
| dc.identifier.doi | 10.6342/NTU201804112 | |
| dc.rights.note | 有償授權 | |
| dc.date.accepted | 2018-09-17 | |
| dc.contributor.author-college | 生命科學院 | zh_TW |
| dc.contributor.author-dept | 生命科學系 | zh_TW |
| 顯示於系所單位： | 生命科學系 | |
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